MKL1在巨核细胞分化中的表达及其作用的实验研究

目的了解MKL1在巨核细胞分化、成熟中的作用。方法以人外周血来源CD34~+细胞巨核细胞分化为模型,采用荧光实时定量PCR法研究MKL1基因在不同分化阶段巨核细胞中的表达情况,通过构建慢病毒载体在CD34~+细胞中过表达MKL1基因,利用血细胞涂片和流式细胞仪考察MKL1过表达细胞经细胞因子刺激分化后的形态、CD41~+百分比及DNA含量。结果经qPCR研究发现,MKL1基因在成熟的巨核细胞中的表达6.8倍高于未分化的CD34~+细胞,以及2倍高于单个核巨核细胞。MKL1过表达组经诱导分化后CD41~+巨核细胞百分比与多倍体细胞百分比分别为61.5%和52.9%,均显著高于对照组36.3%和33.4%的比例。结论MKL1基因在巨核细胞分化中表达逐渐上调,其外源表达促进人外周血来源CD34~+细胞巨核细胞分化和多倍体化。     

Preclinical biology of megakaryocyte growth and development factor: a summary

Since the discovery of the ligand for the cytokine receptor c-Mpl, much has transpired. The development of this protein has been rapid, and the amount of information available on the effects of this molecule in vitro and in vivo is vast. This paper will highlight some of the major studies and observations which are part of the ongoing pre-clinical development of the megakaryocyte growth and development factor encoding the erythropoietin-like domain of the c-Mpl ligand. A summary of in vitro effects on human cells, as well as the key in vivo observations, are included. This molecule is currently in clinical trials, and the initial results are promising.     

Preliminary results of Pneumocystis carinii strain differentiation by using molecular biology.

The mode of Pneumocystis carinii transmission is controversial. Recent studies point to exogenous inoculation rather than reactivation, and person-to-person transmission has also been suggested. Comparison of nucleotide sequences of the large-subunit mitochondrial rRNA gene of P. carinii from human immunodeficiency virus-seropositive patients showed strain differences.     

rhTPO对巨核系的定向分化效应

目的 :探讨重组人促血小板生成素 (rhTPO)对外周造血干细胞中巨核系祖细胞的定向诱导分化能力。方法 :经化疗及G CSF动员后分离的外周造血干细胞 ,进行液体和集落培养 ,研究rhTPO单独及与IL 3、IL 6、SCF的协同作用。结果 :液体培养后 ,TPO组单个核细胞数 (MNC)扩增了 ( 1.73± 0 .49)倍 ,IL 3 IL 6 SCF组MNC比种植时增加 ( 4.2 0± 1.14)倍 ,IL 3 IL 6 SCF TPO组MNC增加了 ( 4.5 3± 1.2 7)倍。单独应用TPO及TPO与IL 3、IL 6、SCF合用产生高比例的CD41a 细胞 ,TPO组培养前CD41a 细胞为 11.70 %± 5 .2 3% ,培养后CD41a 细胞为 19.17%± 6 .2 6 % ( P <0 .0 5 )。CD41a 细胞数在TPO组扩增了 ( 3.5 2± 1.18)倍 ,IL 3 IL 6 SCF组扩增了 ( 5 .32± 1.79)倍 ,IL 3 IL 6 SCF TPO组扩增了 ( 6 .94± 2 .19)倍。结论 :TPO可以定向诱导巨核系细胞的分化 ,IL 3、IL 6、SCF可以协同TPO的作用 ,这在造血调控研究 ,体外定向扩增外周血干细胞 ,促进外周造血干细胞移植患者血小板的恢复具有应用前景     

Characterization, molecular cloning and expression of megakaryocyte potentiating factor

We examined whether the conditioned media of 64 kinds of cell lines, which have been maintained by a protein-free culture system, could produce megakaryocyte potentiating (Meg-POT) activity. In these cell lines, HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from its conditioned medium by a combination of ion-exchange chromatography, gel filtration and reversed-phase HPLC. The purified MPF showed Meg-POT activity almost equal to human (Hu) interleukin 6 (IL-6) in the presence of murine IL-3 in a colony-forming assay with mouse bone marrow cells. The molecular weight of MPF was estimated to be 33 kDa by SDS-PAGE. Glycopeptidase F digestion and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF may be a novel cytokine which has Meg-POT activity. Then, we isolated HuMPF cDNA from an HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The HuMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular weight of 68 kDa, although HPC-Y5 cells secrete a 33 kDa form of HuMPF. HuMPF cDNA does not show any significant homology with other known sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and Meg-POT activity was detected in their culture supernatant. The COS-7 cells secreted only a 33 kDa recombinant (r)HuMPF, however, an additional 30 kDa form was detected in the culture medium of CHO cells. The 33 kDa rHuMPF from CHO cells showed Meg-POT activity, but not the purified 30 kDa rHuMPF. The difference in structure and activity between the 33 and 30 kDa forms of HuMPF was ascribed to the existence in the 33 kDa form of the C-terminal 25 amino acid residues. The expression of MPF mRNA was examined by Northern blot analysis using labeled MPF cDNA as a probe. MPF mRNA was detected in HPC-Y5 cells, with an approximate molecular size of 2.4 kb. We also examined the expression of the MPF gene in various human tissues, and the 2.4 kb band was detected only in lung. Then, the immunohistocytochemical analysis and in situ hybridization revealed that MPF-producing cells were identified as lung macrophages. MPF may exhibit other biological activities such as regeneration of the lung tissues.